Cystic fibrosis, hepatitis, and diabetes are diseases that afflict over 150 million people worldwide. The three above-mentioned diseases have in common that they affect organs derived from the endoderm, one of the three primary layers of cells that compose the human embryo on the tenth day of life, and from which the different body tissues will subsequently develop. In particular, from the endoderm, there will be the generation of: the digestive tract, the respiratory tract, the bladder, part of the urethra and vagina, the middle ear, the mucosa overlying liver and pancreas, the thyroid, the parathyroid glands, and the remaining endocrine glands, the mucosa covering the digestive and respiratory systems.
There are many diseases involving organs derived from the endoderm; for those mentioned above, the current therapies based on organ transplantation are clearly limited by the availability of tissues derived from donors.
An alternative to the organ transplantation is offered by the huge potentialities resulting from the increasingly detailed knowledge of the stem cells, undifferentiated pluripotent cells that proliferate indefinitely and are able to differentiate, potentially giving rise to all the cell types in the body. The pluripotent cells are the embryo cells before the formation of the three germ layers. Said pluripotent stem cells are typically isolated from the inner cell mass of the embryo, at the so-called blastocyst stage. These cells are therefore called embryonic stem cells. An alternative source of pluripotent cells is offered by iPS (induced Pluripotent Stem) cells, which are reprogrammed adult cells and having characteristics similar to the cells that are generated in the embryo, described in JP20050359537.
A crucial step for the achievement of the expected applicative implications of pluripotent cells is to provide methods which allow, in a reproducible way, to induce the differentiation towards the desired cell type. In fact, the application in the regenerative therapy of pluripotent cells becomes a reality when, from said inexhaustible source of cells, it becomes possible to derive precisely the desired cell type. The differentiation must necessarily take place in a homogeneous manner, since the organ replacement is feasible only if the available cell population is composed exclusively of the proper cell type.
In recent years, research made considerable efforts to produce endodermal precursor cells, but the results obtained to date are modest. Among the methods proposed to induce the differentiation of pluripotent cells towards endodermal precursor cells, some authors propose the differentiation through an embryo bodies (EBs) differentiation stage in culture. Through a serum exposure, or due to an exposure to activin A in the absence of serum, the endoderm induction starting from EBs has been shown (Kubo A et al., Development 2004). The critical aspects in said method must necessarily be overcome from a therapeutic point of view. EBs are aggregates of cells derived from embryonic stem cells. Said cells are forced to aggregate by preventing the adhesion thereof to the culture dish bottom.
The thus-aggregated cells begin a differentiation process that somewhat reflects the embryonic development. The obtained differentiation, despite the same happens in a three-dimensional structure, however, is very disorganized when compared to the physiological embryonic development, and the formed three-dimensional structure prevents to highlight and identify all the cells composing it, leading to non-selected cultures in the cell types that compose them. Furthermore, importantly, the three-dimensional structure prevents a uniform exposure of the cells to the factors that are present in the culture medium, and, prospectively, it features greater difficulties when it must be implemented so as to be applicable to large scale processes. Furthermore, the expected serum exposure is to be avoided absolutely from the therapeutic point of view, because the serum is a medium, the chemical composition of which is not necessarily completely defined; furthermore, it is of animal origin, generally bovine origin, thus it can lead to the onset of incompatibility phenomena when cells exposed thereto are transferred into a human being.
A further method for the differentiation towards endoderm is proposed by Kim et al. (Kim PTW et al., PLOS ONE 2010). The authors proceed with the differentiation of embryonic stem cells without the transition to EBs and by using all-trans retinoic acid and dibutyryl cAMP. While overcoming the problems associated to EBs, also in this case there remains the presence of fetal bovine serum in the culture medium.
The induction of endodermal cells starting from ES cells by the use of small molecules capable of crossing the cell membrane is described in Borowiak M et al., Cell Stem Cell 2009. The authors, from a screening of 4,000 molecules, select two molecules which are able to promote the differentiation towards endoderm. The two molecules, so-called IDE1 and IDE2, 2-[(6-carboxy-hexanoyl)-hydrazonomethyl]-benzoic acid and 7-(2-cyclopentylidenehydrazino)-7-oxoheptanoic acid, are putative inhibitors of the histone deacetylase enzymes. In this case also, the culture medium used for the maintenance in culture and the differentiation of ES cells comprises fetal bovine serum.
The object of the present invention is to provide a method which allows obtaining, in an efficient and reproducible manner, and without using components which are not chemically defined in the culture medium, a culture highly enriched with endodermal precursor cells, which cells will be able to find application in the regenerative therapy aimed at diseases involving organs of endodermal derivation.